Phenotypic and Genotypic Characterization of Multidrug-Resistant Enterobacter hormaechei Carrying qnrS Gene Isolated from Chicken Feed in China

ABSTRACT Multidrug resistance (MDR) in Enterobacteriaceae including resistance to quinolones is rising worldwide. The plasmid-mediated quinolone resistance (PMQR) gene qnrS is prevalent in Enterobacteriaceae. However, the qnrS gene is rarely found in Enterobacter hormaechei (E. hormaechei). Here, we reported one multidrug resistant E. hormaechei strain M1 carrying the qnrS1 and blaTEM-1 genes. This study was to analyze the characteristics of MDR E. hormaechei strain M1. The E. hormaechei strain M1 was identified as Enterobacter cloacae complex by biochemical assay and 16S rRNA sequencing. The whole genome was sequenced by the Oxford Nanopore method. Taxonomy of the E. hormaechei was based on multilocus sequence typing (MLST). The qnrS with the other antibiotic resistance genes were coexisted on IncF plasmid (pM1). Besides, the virulence factors associated with pathogenicity were also located on pM1. The qnrS1 gene was located between insertion element IS2A (upstream) and transposition element ISKra4 (downstream). The comparison result of IncF plasmids revealed that they had a common plasmid backbone. Susceptibility experiment revealed that the E. hormaechei M1 showed extensive resistance to the clinical antimicrobials. The conjugation transfer was performed by filter membrane incubation method. The competition and plasmid stability assays suggested the host bacteria carrying qnrS had an energy burden. As far as we know, this is the first report that E. hormaechei carrying qnrS was isolated from chicken feed. The chicken feed and poultry products could serve as a vehicle for these MDR bacteria, which could transfer between animals and humans through the food chain. We need to pay close attention to the epidemiology of E. hormaechei and prevent their further dissemination. IMPORTANCE Enterobacter hormaechei is an opportunistic pathogen. It can cause infections in humans and animals. Plasmid-mediated quinolone resistance (PMQR) gene qnrS can be transferred intergenus, which is leading to increase the quinolone resistance levels in Enterobacteriaceae. Chicken feed could serve as a vehicle for the MDR E. hormaechei. Therefore, antibiotic-resistance genes (ARGs) might be transferred to the intestinal flora after entering the gastrointestinal tract with the feed. Furthermore, antibiotic-resistant bacteria (ARB) were also excreted into environment with feces, posing a huge threat to public health. This requires us to monitor the ARB and antibiotic-resistant plasmids in the feed. Here, we demonstrated the characteristics of one MDR E. hormaechei isolate from chicken feed. The plasmid carrying the qnrS gene is a conjugative plasmid with transferability. The presence of plasmid carrying antibiotic-resistance genes requires the maintenance of antibiotic pressure. In addition, the E. hormaechei M1 belonged to new sequence type (ST). These data show the MDR E. hormaechei M1 is a novel strain that requires our further research.

L55 -change "caused" to "causes" L56 -insert "a" before "green" L57-59 -sentence does not make sense, please re-write. L59 -delete "gene" L60 -insert "a" before "protein" L61 -quinolones should be singular L61 -split paragraph before "PMQR" L65 -correct spelling of "homaechei" L68-81 -this is a summary of the work conducted in this report and it should be deleted because it is redundant with the M&M section. Instead, the objectives of the study should be briefly stated. L85 -insert a space in "4ml" and change "ml" to "mL" to be consistent with the rest of the manuscript. L86 -delete "the feed" L102 -"Molecular" does not need to be capitalized L104 -change "expanding" to "propagating" L121-128 -is this the list of the 17 strains? What do you mean by "were Homo sapiens"? L129 -how many times the susceptibility tests were repeated? L136 -insert "strain" before "was" L139 -delete "The" L140 -replace "the MacConkey plates were supplemented" with "and" L142 -delete first "strain" after "donor" L143 -delete "an" L145 -insert a space in "0.22µm" L149 -"proceed" -did you mean "processed"? L166 -insert a space in "2µg" L191 -Result should be plural L234 -insert "was sensitive" before "to" L235 -delete "was sensitive" L242-243 -what do you mean "but also emerged corresponding resistance" L252 -revise sentence "passage" is a noun not a verb L256 -revise sentence, poor grammar. L258 -delete "mainly" it is unnecessary L258-263 -revise paragraph, only the first two sentences are related. The last sentence is not necessary a conclusion from the previous 3 sentences. It needs better logic. L264-a single detection on a chicken feed sample can not lead to the conclusion that it is an emergence. Because there is no information about the representativeness of the feed sample, all that can be concluded is that this is the first isolation from chicken feed. L337 -revise sentence, poor grammar. Reference section -needs to be thoroughly edited to comply with a consistent referencing format. All bacterial names are not italicized. Table 1 -the results of a positive or standard E. hormaechi are missing in this table and in its experimental design. This study by Cao et al, summarizes the Phenotypic and genotypic characterization of a MDR Enterobacter hormaechei isolate carrying qnrS gene isolated from chicken feed. While this study sheds some light into the emergence of Plasmid-mediated quinolone resistance (PMQR) genes in newer Enterobacter spp. isolated from Cattle feeds, it falls short in demonstrating the linkage between the origins of the PMQR in Chicken Feed and their spread in Chickens.

Introduction
Lines 64-65-However, there are few reports on the prevalence of qnrS in E. homachei. No Reference has been provided for this statement. In fact, the authors have failed to quote/refer/acknowledge another recently published study by Ai et al, reporting the coexistence of qnr genes in a MDR E. hormaechei isolated from humans. (https://www.frontiersin.org/articles/10.3389/fmicb.2021.676113/full) Lines 66-81-This portion sounds like a rehash of their Abstract, with a summary of their Abstract/results (verbatim from the Abstract on several instances). Requires extensive rewriting (with minimal repetition) to setup the context of this study in this section.

Methodology
Line-87-Why a differential media such as EMB/MaConkey agar plates were not used here (in additon to a general media-LB agar)?
Line 92-94-Why were Antibiotics purchased from a private entity (Dr. Ehrenstorfer (Germany)) and not a commercial vendor following standard Quality procedures.
Line 126-"BD-50-Eh (CP063224.1) were Homo sapiens"-This section is not clear-Unable to deduce what the authors are trying to convey here.
Line 147-"which were equably dripped on the filter membrane"-could be written as "which were equably aliquoted onto the filter membrane" Inconsistent "verb tense" throughout the methodlogy section, that swiches back and forth.

Results
Sections 3-1 & 3.2-The authors have sequenced the Whole genome and Plasmids of Enterobacter hormaechei, but have not provided a Genbank ID for the sequences obtained from this study in these sections (unless I have overlooked). Section 3.3-A table with a list of "Unique genes in pM1" (as compared to other plasmids in Fig 1) would be helpful for easier Interpretation of the data.
Lines 216-217-Were there sequence evidence for a Operon? If so, did the authors try using a PCR for amplifying this Operon region among Transconjugants?
Lines 282-284-Consider rephrasing this sentence-"In this research also found IS407 and IS2A insertion elements located upstream of blaTEM-1 and qnrS1 genes. Insertion elements upstream of genes affect antibiotic resistance phenotypes (44)" "Chicken Feed" being the epicenter of this study, No discussion points have been made or reference(s) provided to discuss the origins of the PMQR in Chicken Feed?
Staff Comments:

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Many of the recommendations were incorporated, despite the authors assurances, several important changes were not actually performed. L59 -sentence was not modified as suggested in the previous version for L52 L77-84 -paragraph was modified, but it still includes a summary of the work performed. It is inappropriate for an introduction. Please delete and just state the objectives of the work.
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